Polyvinyl alcohol microspheres, injectable solutions and therapeutic uses of the same

ABSTRACT

The present invention relates to microspheres useful for embolization which comprises polyvinylalcohol. The present invention also relates to an injectable suspension suitable for embolization which comprises the polyvinylalcohol microspheres and a suitable liquid carrier. The present invention further relates to a method for prophylactic or therapeutic embolization which comprises administering to a mammal an injectable suspension containing the polyvinylalcohol microspheres and a suitable liquid carrier. Finally, the present invention relates to a process for producing the polyvinylalcohol microspheres.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 12/348,867filed Jan. 5, 2009, which is a divisional application of U.S. Ser. No.10/692,785 (U.S. Pat. No. 7,591,993) filed Oct. 27, 2003, which is adivisional application of U.S. Ser. No. 09/419,114 (U.S. Pat. No.6,680,046) filed Oct. 15, 1999, which claims priority to Franceapplication Serial No. 98-13019 filed Oct. 16, 1998, each of which isherein incorporated by reference in its entirety.

1. FIELD OF INVENTION

The present invention relates to materials useful for embolization,methods for using the same for embolization and processes for producingsuch materials.

2. BACKGROUND OF THE INVENTION

Therapeutic vascular occlusions (embolizations) are used to prevent ortreat certain pathological conditions in situ. Generally they areemployed using catheters, under imagery control, to position particulateocclusion agents (emboli) in the circulatory system. Embolizations canbe used in a variety of vessels and organs whether healthy or diseased;however, they are more commonly used in conditions such as, e.g.,tumors, vascular malformations, hemorrhagic processes, etc. Notably, inthe case of tumors, vascular occlusion can suppress pain, limit bloodloss during surgical intervention following embolization or even bringon tumoral necrosis and avoid the necessity for surgical intervention.In the case of vascular malformations, embolization enables the bloodflow to the “normal” tissues to be normalized, aids in surgery andlimits the risk of hemorrhage. In hemorrhagic events or processes,vascular occlusion produces a reduction of blood flow, which promotescicatrization of the arterial opening(s).

Furthermore, depending on the pathological conditions treated,embolization can be used for temporary as well as permanent objectives.

Embolization has been performed with a variety of solid materials suchas small pieces of dura mater, irregular polyvinylalcohol particles,irregular gelatin particles, and more recently with crosslinkedspherical hydrogel made from a polyacrylamide derivative and acrosslinked gelatin.

U.S. Pat. No. 5,635,215 discloses microspheres, comprising a hydrophilicacrylic copolymer coated with a cell adhesion promoter and a markingagent which are useful for embolization. U.S. Pat. No. 5,648,100discloses an injectable solution for therapeutic embolization,comprising microspheres comprising a hydrophilic acrylic copolymercoated with a cell adhesion promoter and a marking agent. U.S. Pat. No.5,648,100 also discloses a method for therapeutic embolization whichcomprises administering to a mammal the above injectable solution.

The most common material used to date in a variety of embolizationapplications is irregular polyvinylalcohol particles. However, theseirregular polyvinylalcohol particles have numerous drawbacks, and can incertain circumstances even led to deaths. For example, Repa et al.,Radiology, 1987, 170:395-399 discloses that two infants with symptomatichepatic arteriovenous malformation (AVM) were treated with catheterembolization using commercially available polyvinylalcohol (IVALONparticle suspensions from Laboratory Ingenor (Paris)). Both infants diedsoon after the AVM embolization. Further examination demonstrates thatmarked heterogeneity of particle size very probably contributed to thedeath of the infants. Indeed, these and other problems are associatedwith irregular polyvinylalcohol particles mostly due to their particleshapes. These problems make it difficult, or even dangerous in certaincases, to use irregular polyvinylalcohol particles in embolization.

Polyvinylalcohol products are commercially available from TargetTherapeutics/Boston Scientific (CONTOUR), from Nycomed (IVALON,ULTRA-DRIVALON, and ULTRA-IVALON), from Cordis (TRUFILL) and from Cook(PVA). These polyvinylalcohol particles are known to be irregularlyshaped particles. Generally, these polyvinylalcohol particles are soldas dry powders or saline suspensions. Despite their potential damage,irregular polyvinylalcohol particles have been used extensively.Examples of the use of irregular polyvinylalcohol particles arediscussed below.

Kusano et al., Invest. Radiol., 1987, 22:388-392, discloses low-doseparticulate polyvinylalcohol embolization in animal and clinicalstudies. Polyvinylalcohol particles used in Kusano were IVALON obtainedfrom Unipoint Laboratory, High Point, N.C., in the radiopaque form.Kusano discloses that low-dose large polyvinylalcohol particles(diameter at 590-1000 μm) are suitable as an embolic material fortrans-catheter occlusion of small intestinal hemorrhage in patients withcertain diseases such as stress ulcer, surgical drain, anastomosis,tuberculous ulcer and nonspecific ulcer.

Rump et al., Gen. Pharmac., 1996, 27(4):669-671, disclosespharmacokinetics of intraarterial Mitomycin C (MMC) in thechemo-embolization treatment of liver metastases. In Rump, hepaticbranches of patients with primary colorectal cancer and liver metastaseswere embolized using irregular polyvinylalcohol particles (150-250 μm)before applying MMC.

Barton et al., JVIR, 1996, 7:81-88, discloses embolization of patientswith bone metastases to prevent major blood loss during surgery, toreduce bone metastases, to reduce pain and to control heavy bleeding.Polyvinylalcohol foam particles (IVALON; DRIVALON 300-600 μm;Nycomed-Ingenor, Paris) were used in eight cases in Barton.

Wakhloo et al., AJNR, 1993, 14:571-582, discloses extended preoperativemicro-embolization of intracranial meningiomas using 50-150 μm and150-300 μm polyvinylalcohol particles. Wakhloo concluded from theirstudy that embolization with 50-150 μm irregular polyvinylalcoholparticles led to a higher percentage of effective tumordevascularization and tumor necrosis for intracranial meningiomas.

Given the interest in the use of polyvinylalcohol particles forembolization, there is a great need for a safe and effective method forits application. The present invention addresses these and other needsin the art.

3. SUMMARY OF THE INVENTION

Despite the risks and difficulties associated with the use ofpolyvinylalcohol particles in embolization, applicant has discoveredsurprisingly that microspheres made from crosslinked polyvinylalcoholare biocompatible, non-toxic and safe in embolization procedures.Accordingly, the present invention encompasses microspheres useful forembolization which comprise crosslinked polyvinylalcohol microspheres,injectable suspensions suitable for embolization which comprise thecrosslinked polyvinylalcohol microspheres and a suitable liquid carrier,methods for prophylactic or therapeutic embolization using suchinjectable suspensions, and processes for producing the crosslinkedpolyvinylalcohol microspheres.

The invention described herein encompasses microspheres, havingdiameters ranging from about 10 μm to about 2,000 μm useful forembolization, which comprise crosslinked polyvinylalcohol. Themicrospheres of the present invention can be in the form of dry powderor hydrogel. In one embodiment, the present invention encompassesmicrospheres which comprise, in crosslinked and hydrogel form, about0.5% to about 20% polyvinylalcohol by weight. In another embodiment, thepresent invention encompasses crosslinked polyvinylalcohol microsphereswhich further comprise a cell adhesion promoter, a marking agent, orboth. In still another embodiment, the present invention encompassespolyvinylalcohol microspheres further comprising an anti-angiogenicagent.

The present invention also encompasses an injectable suspension suitablefor prophylactic or therapeutic embolization, which comprisesmicrospheres, having diameters ranging from about 10 μm to about 2,000μm which comprise crosslinked polyvinylalcohol and a suitable liquidcarrier. In a preferred embodiment, the present invention encompasses aninjectable suspension wherein the microspheres comprise, in crosslinkedand hydrogel form, about 0.5% to about 20% polyvinylalcohol by weight.In one embodiment, the microspheres in said injectable suspension have auniform or narrow size range, wherein the difference in diameter betweenthe microspheres is from about 0 μm to about 150 μm, preferably fromabout 0 μm to about 100 μm. In another embodiment, the present inventionencompasses an injectable suspension wherein the crosslinkedpolyvinylalcohol microspheres further comprise a cell adhesion promoter,a marking agent or both. In still another embodiment, the presentinvention encompasses an injectable suspension wherein thepolyvinylalcohol microspheres further comprise an anti-angiogenic agent.

The present invention additionally encompasses a method for prophylacticor therapeutic embolization in a mammal which comprises administering tosaid mammal an injectable suspension comprising an effective amount ofmicrospheres, having diameters ranging from about 10 μm to about 2,000μm, which comprise crosslinked polyvinylalcohol. An effective amount ofsaid microspheres is generally the amount sufficient to occlude thevessel in question. In general, this amount is between a few dozen to afew hundred microspheres. In a preferred embodiment, the presentinvention encompasses a method for embolization wherein the crosslinkedpolyvinylalcohol microspheres being administered in the injectablesuspension comprise from about 0.5% to about 20% crosslinkedpolyvinylalcohol by weight in the hydrogel form. In another embodiment,the present invention encompasses a method for embolization wherein thecrosslinked polyvinylalcohol microspheres being administered furthercomprise a cell adhesion promoter, a marking agent, or both. In stillanother embodiment, the present invention encompasses a method forembolization wherein the polyvinylalcohol microspheres beingadministered further comprise an anti-angiogenic agent.

The present invention further encompasses a process for producingcrosslinked polyvinylalcohol microspheres, having a diameter rangingfrom about 10 μm to about 2,000 μm, which comprises: a) dissolvingpolyvinylalcohol in an acidic solution; b) adding an aldehyde to saidpolyvinylalcohol-containing solution, or vice verse, to form a mixture;c) adding said mixture, with agitation, to an oil containing from about0.1% to about 10% of an emulsifier having Hydrophilic-HydrophobicBalance (“HLB”) less than 5, or vice verse, to form an emulsion withdroplets of polyvinylalcohol suspended in said oil; d) heating saidemulsion to condense said aldehyde on polyvinylalcohol chains andthereby forming spherical particles of crosslinked polyvinylalcohol; e)removing said oil from said spherical particles of crosslinkedpolyvinylalcohol; f) neutralizing said active aldehyde on said sphericalparticles of crosslinked polyvinylalcohol; g) washing said neutralizedspherical particles of crosslinked polyvinylalcohol with physiologicalaqueous buffers; and preferably h) sterilizing said washed sphericalparticles of crosslinked polyvinylalcohol. Thepolyvinylalcohol-containing solution used in this process preferable hasa polyvinylalcohol concentration from about 0.5% to about 20% (w/v).

4. DETAILED DESCRIPTION OF THE INVENTION

Microspheres useful for embolization which comprise polyvinylalcohol,injectable suspensions suitable for embolization which comprise thepolyvinylalcohol microspheres, methods for prophylactic or therapeuticembolization using such injectable suspensions, and processes forproducing the polyvinylalcohol microspheres are described herein.

As used herein, “microspheres” means solid insoluble particles which maybe suspended in biological or biologically-compatible liquids, and whichhave, under microscopic examination, substantially a sphere or aspheroidal shape (ellipsis). A sphere is defined as a volume thatpresents the lowest external surface area. The surface of microspheresappear smooth under less than 1000-fold magnifications.

As used herein, “irregular particles” means solid insoluble particles,under microscopic examination, have a shape that is not a substantiallysphere or spheroidal (ellipsis). The shape of irregular particles isoften the result of a larger solid particle that has been crushed. Eachirregular particle appears non-uniform in shape as compared tomicrospheres. Also in contrast to microspheres, irregular particles haverough surface. The length, thickness and depth of irregular particlesare not uniform; they show angles and protuberances on the surface.These particles also appear irregular in their ability to transmit lightunder microscopic examination, depending on the thickness of theparticles at particular locations.

The use of irregular particles in embolization has certain drawbacks.First, spheres are defined by their diameter. Irregular particles cannot be defined geometrically except by their whole volume and do nothave real dimensions. Therefore, irregular particles can not beaccurately sieved to achieve a uniform or even narrow range sizedistribution. As a result, it is difficult to properly and completelyocclude artery lumen using irregular particles because they can notestablish complete contact with all the surface of the artery which iscylindrical. In addition, irregular particles sometimes block thecatheter lumen depending on their space orientation inside the lumen ofa catheter. Moreover, as a result of the rough surface of irregularparticles and the possibility that such particles may break as aconsequence of attrition phenomena, very small-sized particles can begenerated from the irregular particles. When such very small-sizedparticles are generated during handling or administration in vivo,inadvertent pulmonary embolization, a potentially fatal complication,can occur. Furthermore, irregular particles have large surface area incomparison to their volume. They tend to form clumps or aggregations,which are responsible for catheter clogging and undesired proximalembolization.

In contrast, use of microspheres described herein in embolization hascertain advantages. For example, due to their spherical shape orsubstantially spherical shape, microspheres can properly and completelyocclude artery lumen because they can establish complete contact withall the surface of the artery which is cylindrical. In addition, themicrospheres of the present invention can be easily calibrated, andsamples or suspensions containing these microspheres will not block orclog catheters because they always have the same dimension regardless oftheir space orientation in the catheter. Moreover, due to their smoothsurface, no attrition will occur and small-sized particles will not begenerated from the microspheres; thus avoiding the potentially fatalcomplications, such as pulmonary embolization. Furthermore, microspherescan only interact with each other on a single point and such contact isnot enough to induce aggregation by surface interaction.

The invention described herein encompasses microspheres, having adiameter ranging from about 10 μm to about 2,000 μm, useful forembolization which comprises crosslinked polyvinylalcohol. Preferreddiameters for the present invention will depend on the type ofembolization and can be readily determined by the skilled artisans. Themicrospheres of the present invention can be in the form of dry powderor hydrogel. In a preferred embodiment, the present inventionencompasses microspheres, which comprise in crosslinked and hydrogelform, from about 0.5% to about 20% crosslinked polyvinylalcohol byweight. In other embodiments, the crosslinked polyvinylalcoholmicrospheres may further comprise one or more of a cell adhesionpromoter, a marking agent, or an anti-angiogenic agent.

The present invention also encompasses an injectable suspension suitablefor embolization, which comprises crosslinked polyvinylalcoholmicrospheres, having a diameter ranging from about 10 μm to about 2,000μm and a suitable liquid carrier. In a preferred embodiment, thecrosslinked polyvinylalcohol microspheres in said injectable suspensionhave a uniform or narrow size range, wherein the difference in diameterbetween the microspheres is from about 0 μm to about 150 μm, preferablyfrom about 0 μm to about 100 μm. In other embodiments, the presentinvention encompasses an injectable suspension wherein the microspheresare comprised of from about 0.5% to about 20% crosslinkedpolyvinylalcohol by weight in the hydrogel form; an injectablesuspension wherein the crosslinked polyvinylalcohol microspheres mayfurther comprise a cell adhesion promoter, a marking agent, and aninjectable solution wherein the polyvinylalcohol microspheres and ananti-angiogenic agent.

The present invention additionally encompasses a method for prophylacticor therapeutic embolization in a mammal which comprises administering tosaid mammal in need of such embolization an injectable suspensioncomprising an effective amount of crosslinked polyvinylalcoholmicrospheres, having diameters ranging from about 10 μm to about 2,000μm, and a suitable liquid carrier. In a preferred embodiment, thepresent invention encompasses a method for therapeutic embolizationwherein the polyvinylalcohol microspheres in the injectable suspensionbeing administered comprise from about 0.5% to about 20% crosslinkedpolyvinylalcohol by weight in the hydrogel form. In other embodiments,the crosslinked polyvinylalcohol microspheres being administered in saidmethod for prophylactic or therapeutic embolization may further compriseone or more of a cell adhesion promoter, a marking agent and ananti-angiogenic agent.

The present invention further encompasses a process for producingcrosslinked polyvinylalcohol microspheres, having diameters ranging fromabout 10 μm to about 2,000 μm. Various acidic solutions, aldehydes,oils, emulsifiers, agitation speeds, heating conditions and oil removingmethods can be used in the process as described below. In otherembodiments, the present invention encompasses a process for producingcrosslinked polyvinylalcohol microspheres further comprising adding acell adhesion promoter to the acidic polyvinylalcohol solution beforeadding the aldehyde; a process further comprising absorbing a markingagent into the crosslinked polyvinylalcohol microspheres; and a processfurther comprising absorbing an anti-angiogenic agent into thecrosslinked polyvinylalcohol microspheres.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the present invention is divided into the subsectionswhich follow.

4.1. Polyvinylalcohol Microspheres

Polyvinylalcohol is a polymer prepared from polyvinyl acetates byreplacement of the acetate groups with hydroxyl groups. Examples ofother names for polyvinylalcohol include, but are not limited to, AkwaTears, Elvanol, Gelvatol, Lipuifilm, Mowiol, Polyviol, Sno Tears,Vinarol and Vinol (The Merck Index, 12th Ed., Merck & Co., Inc., 1996, p1308). Such synonyms are encompassed by the present invention.Polyvinylalcohol can be synthesized according to the proceduresdisclosed in Hermann, Haehnel, Ber. 60:1658 (1927); Schildknecht, Vinyland Related Polymers (Wiley, New York, 1952); Staudinger et al., Ber.60:1782 (1927); Prakt, Chem., 155:261 (1940); Marvel, J. Am. Soc.,60:1045 (1938); McDowell, J. Am. Soc., 62:415 (1940); Marvel, J. Am.Soc., 65:1710 (1943); Leeds, Encyclopedia of Chemical Technology(KirkOthmer ed.), 21:353-368 (Wiley-Interscience, New York, 2nd ed.,1970); Polyvinyl Alcohol (Finch Ed.), p 640 (Wiley, New York, 1973); andDunn, Chem & Ind. (London), pp 801-806 (1980). Polyvinylalcohol can alsobe obtained from commercial chemical suppliers such as Aldrich, Flukaand Sigma.

The present invention provides polyvinylalcohol microspheres having oneor more of the following characteristics: 1) substantially spherical; 2)substantially uniform in size and shape; 3) will not aggregate bysurface interaction; and 4) the diameter of which can easily becalibrated.

Polyvinylalcohol microspheres having a diameter ranging from about 10 μmto about 2,000 μm are also provided in the present invention. Themicrospheres of the present invention can be in the form of dry powderor hydrogel. In one embodiment, crosslinked hydrogel microspheres of thepresent invention comprise about 0.5% to about 20% crosslinkedpolyvinylalcohol by weight.

The present invention also provides crosslinked polyvinylalcoholmicrospheres which further comprise a cell adhesion promoter, a markingagent or both. Such cell adhesion promoter include, but are not limitedto, CM dextran, collagen, DEAE dextran, gelatin, glucosaminoglycans,fibronectin, lectins, polycations, natural biological cell adhesionagents or synthetic biological cell adhesion agents. In a preferredembodiment, the cell adhesion promoter is selected from the groupconsisting of CM dextran, collagen and DEAE dextran.

The marking agents useful within the present invention include, but arenot limited to, dyes, imaging agents and contrasting agents. Examples ofchemical dyes that can be used in the present invention, which makepossible a direct visualization of the microspheres, include, but arenot limited to, Cibacron Blue and Procion Red HE-3B. Examples of imagingagents that can be used in the present invention include, but are notlimited to, magnetic resonance imaging agents such as erbium, gadoliniumand magnetite. In a preferred embodiment, a magnetite imaging agent,such as ferrofluid, is used. Examples of contrasting agents that can beused in the present invention include, but are not limited to, barium oriodine salts and amino-3-triiodo-2,4,6-benzoic acid. The use andpreparation of the above dyes, imaging agents and contrasting agents aredisclosed in U.S. Pat. Nos. 5,635,215; 5,648,100; Boschetti,Biochem-Biophys. Meth. 19: 21-36 (1989); and Boschetti et al., Bull.Sec. Chim. France. 1986 No. 4), the contents of which are incorporatedherein by reference.

In the case of barium or magnetite salts, they can be directlyintroduced in powdered form in the initial polyvinylalcohol solution inthe process of preparing polyvinylalcohol microspheres according to thepresent invention. It is also possible to incorporate such markingagents into the microspheres after their synthesis. This can be done,for example, by grafting of fluorescent markers such as erythrosine orfluorescein or their derivatives (FITC, EITC, and the like).

In another embodiment, the present invention provides crosslinkedpolyvinylalcohol microspheres further comprising an anti-angiogenicagent.

The anti-angiogenic agents useful within the present invention include,but are not limited to, AGM-1470 (TNP-470), angiostatic steroids,angiostatin, antibodies against avβ3, antibodies against bFGF,antibodies against IL-1, antibodies against TNF-α, antibodies againstVEGF, auranofin, azathioprine, BB-94 and BB-2516, basic FGF-solublereceptor, carboxyamido-trizole (CAI), cartilage-derived inhibitor (CDT),chitin, chioroquine, CM 101, cortisone/heparin, cortisone/hyaluroflan,cortexolone/heparin, CT-2584, cyclophosphamide, cyclosporin A,dexamethasone, diclofenac/hyaluronan, eosinophilic major basic protein,fibronectin peptides, Glioma-derived angiogenesis inhibitory factor(GD-AIF), GM 1474, gold chloride, gold thiomalate, heparinases,hyaluronan (high and low molecular-weight species),hydrocortisonelbeta-cyclodextran, ibuprofen, indomethacin,interferon-alpha, interferon gamma-inducible protein 10,interferon-gamma, IL-1, IL-2, IL-4, IL-12, laminin, levamisole,linomide, LM609, martmastat (BB-2516), medroxyprogesterone,methotrexate, minocycline, nitric oxide, octreotide (somatostatinanalogue), D-penicillamine, pentosan polysulfate, placentalproliferin-related protein, placental RNase inhibitor, plasminogenactivator inhibitor (PAIs), platelet factor-4 (PF4), prednisolone,prolactin (16-kDa fragment), proliferin-related protein, prostaglandinsynthase inhibitor, protamine, retinoids, somatostatin, substance P,suramin, SU1O1, tecogalan sodium (05-4152),tetrahydrocortisolsthrombospondins (TSPs), tissue inhibitor ofmetalloproteinases (TIMP 1, 2, 3), thalidomide, 3-aminothalidomide,3-hydroxythalidomide, metabolites or hydrolysis products of thalidomide,3-aminothalidomide, 3-hydroxythalidomide, vitamin A and vitreous fluids.In another preferred embodiment, the anti-angiogenic agent is selectedfrom the group consisting of thalidomide, 3-aminothalidomide,3-hydroxythalidomide and metabolites or hydrolysis products ofthalidomide, 3-aminothalidomide, 3-hydroxythalidomide. In a preferredembodiment, the anti-angiogenic agent is thalidomide. The aboveanti-angiogenic agents are disclosed in U.S. Pat. Nos. 5,593,990;5,629,327; and 5,712,291; Norrby, APMIS, 1997, 105:417-437; O'Reilly,Investigational New Drugs, 1997, 15:5-13; and J. Nat'l Cancer Inst.,1996, 88(12):786-788, the contents of which are incorporated herein byreference.

The crosslinked polyvinylalcohol microspheres of the present inventioncan be stored and maintained in the form of dry powders, or as hydrogelsuspended in a suitable liquid carrier.

4.2. Injectable Suspensions Comprising Polyvinylalcohol Microspheres

The present invention provides an injectable suspension suitable forembolization, which comprises microspheres, having diameters rangingfrom about 10 μm to about 2,000 μm, useful for embolization, and asuitable carrier. Preferably, the injectable suspension is sterile.

The various specific and preferred polyvinylalcohol microspheres thatare described in §4.1. can be used in the injectable suspension.

Kits containing a ready made injectable suspension, or thepolyvinylalcohol microspheres described in §4.1. above in powder form,and physiologically acceptable carrier liquid(s) or solution(s) that cansolubilize the polyvinylalcohol microspheres powders, are includedwithin the present invention. Suitable liquid carriers for use in theinjectable suspensions of the present invention include biologicalliquids or solutions and liquids or solutions which are biologicallycompatible or physiologically acceptable. Examples of such liquids orsolutions include, but are not limited to, aqueous solutions, saline,physiological solutions which contain sugars, and the like. Such kitscan also contain cell adhesion promoters, marking agents, oranti-angiogenic agents, or mixtures thereof. Such kits can furthercontain injection means such as a needle, a catheter, guides, contrastagents, and physiological dyes, such as methylene blue.

4.3. Methods for Embolization Using the Injectable SuspensionsComprising Polyvinylalcohol Microspheres

The present invention provides a method for prophylactic or therapeutic,transient or permanent, embolization in a mammal which comprisesadministering to said mammal in need of such embolization an injectablesuspension comprising an effective amount of microspheres, havingdiameters ranging from about 10 μm to about 2,000 μm, useful forembolization, wherein said microspheres comprise crosslinkedpolyvinylalcohol. In a preferred embodiment, the mammal being embolizedis a human.

The various specific and preferred injectable suspensions comprising thepolyvinylalcohol microspheres that are described in §4.1 and §4.2 can beused in the embolization methods of the present invention.

Conditions and disease states that can be prevented or treated by thepresent embolization methods include, but are not limited to, solidtumors, vascular malformations, and hemorrhagic events or processes.Regarding tumors, the present embolization methods can be used tosuppress pain, to limit blood loss occurring during surgicalintervention following embolization, or to bring on tumoral necrosis andto either avoid or minimize the necessity of surgical intervention. Withrespect to vascular malformations, the present embolization methods canbe used to normalize the blood flow to “normal” tissues, to aid insurgery and to limit the risk of hemorrhage. For hemorrhagic events orprocesses, the present embolization methods can be used to reduce bloodflow and to promote cicatrization of the arterial opening(s). Inaddition, the present embolization methods can be used as a pre-surgicaltreatment in order to decrease the blood flow in blood rich organs(e.g., the liver) prior to surgical intervention. Examples of specificconditions that can be prevented or treated by the present embolizationmethods include, but are not limited to: uterine tumors or fibroids;small intestinal hemorrhage, such as that associated with stress ulcer;surgical drain; anastomosis; tuberculous ulcer and nonspecific ulcer;symptomatic hepatic arteriovenous malformation (AVM); primary colorectalcancer; hepatocellular carcinomas; liver metastases; bone metastases;melanomas; cancers of the head or neck; and intracranial meningiomas.

The magnitude of a prophylactic or therapeutic dose of thepolyvinylalcohol microspheres of the present invention, of course, varywith the nature of the type, location and severity of the condition tobe treated and the route of administration. It will also vary accordingto the age, weight and response of the individual patient. Effectiveamounts of the polyvinylalcohol microspheres to be used in theembolization methods of the present invention are based on therecommended doses known to those skilled in the art for the variousconditions, diseases or disorders.

An effective amount refers to that amount of polyvinylalcoholmicrospheres sufficient to result in amelioration of symptoms or aprolongation of survival in a patient. Toxicity and therapeutic efficacyof such polyvinylalcohol microspheres can be determined by standardembolization procedures in experimental animals, or that is sufficientto permanently or temporarily occlude the vascular lumen in question.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of polyvinylalcohol microspheres of thepresent invention at the desired target or location. For example,parenteral, subcutaneous, intramuscular, and the like may be employed. Apreferred mode of administration is delivery inside targeted arteriesvia a catheter.

4.4. Processes for Producing Polyvinylalcohol Microspheres

The present invention provides a process for producing crosslinkedpolyvinylalcohol microspheres, having a diameter ranging from about 10μm to about 2,000 μm, which comprises: a) dissolving polyvinylalcohol inan acidic solution; b) adding an aldehyde to saidpolyvinylalcohol-containing solution to form a mixture, or vice verse;c) adding said mixture, with agitation, to an oil containing from about0.1% to about 10% of an emulsifier having HLB less than 5, or viceverse, to form an emulsion with droplets of polyvinylalcohol suspendedin said oil; d) heating said emulsion to condense said aldehyde onpolyvinylalcohol chains and thereby forming spherical particles ofcrosslinked polyvinylalcohol; e) removing said oil from said sphericalparticles of crosslinked polyvinylalcohol; f) neutralizing said activealdehyde on said spherical particles of crosslinked polyvinylalcohol; g)washing said neutralized spherical particles of crosslinkedpolyvinylalcohol with physiological aqueous buffers; and optionally h)sterilizing said washed spherical particles of crosslinkedpolyvinylalcohol. Various acidic solutions, aldehydes, amino-containingagents, oils, emulsifiers, agitation speeds, heating conditions and oilremoving methods can be used in the process.

Various preferred reagents and reaction conditions can be used in theprocess for producing crosslinked polyvinylalcohol microspheres, asskilled artisans will be aware. For example, in step (a), preferredacidic solutions are 0.5 M H₂SO₄—NaCl and 1 M HCl. In step (b), thepreferred aldehyde is selected from the group consisting offormaldehyde, glyoxal, glutaraldehyde and terephalaldehyde. Morepreferably, the aldehyde is glutaraldehyde. In step (c): 1) thepreferred oil is selected from the group consisting of vegetal oils(e.g., olive oil, corn oil and sunflower oil), mineral oils (e.g.,paraffin oil and silicone oil) and non-polar solvents, and morepreferably, the oil is a mineral oil such as paraffin oil; and thepreferred emulsifier having HLB less than 5 are preferably used inconcentrations from about 0.05% to 5%, and can be selected from thegroup consisting of sorbitan sesquioleate, sorbitan trioleate, sorbitantristearate, polyethylene sorbitan monostearate, cellulose acetatebutyrate and tetradecanol. The agitation speed used in the process ofthe present invention will depend upon type of agitation equipment beingused and the desired size for the microspheres being produced. In step(d) the heating is preferably conducted at about 80° C. for about 6hours. In step (e) said oil is removed from said spherical particles ofcrosslinked polyvinylalcohol using extraction agents such as lightnon-polar solvents, chlorinated solvents, ethyl-ether, and supercriticalcarbon dioxide, and preferably by extraction with light non-polarsolvent or chlorinated solvent, and more preferably, by extraction withmethylene chloride. In step (f) said active aldehyde on said sphericalparticles of crosslinked polyvinylalcohol is preferably neutralized byan amino-containing agent, such as aminoalcohols, e.g., Tris,2-aminoethanol, aminosorbitol and glucosamine, and more preferably, by a0.5 M Tris-HCl buffer (pH 9).

In still another embodiment, the present invention provides a processfor producing crosslinked polyvinylalcohol microspheres furthercomprising adding a cell adhesion promoter to the acidicpolyvinylalcohol solution before adding the aldehyde. In a preferredembodiment, the cell adhesion promoter is selected from the groupconsisting of CM dextran, collagen, DEAE dextran, gelatin,glucosaminoglycans, fibronectin, lectins, polycations, naturalbiological cell adhesion agents or synthetic biological cell adhesionagents. In a more preferred embodiment, the cell adhesion promoter isselected from the group consisting of CM dextran, collagen, and DEAEdextran.

In another embodiment, the present invention provides a process forproducing crosslinked polyvinylalcohol microspheres further comprisingabsorbing a marking agent into the crosslinked polyvinylalcoholmicrospheres. Preferably, the marking agent is selected from the groupconsisting of a dye, an imaging agent and a contrasting agent, and morepreferably, the marking agent is an imaging agent such as ferrofluid.

In still another embodiment, the present invention provides a processfor producing crosslinked polyvinylalcohol microspheres furthercomprising absorbing an anti-angiogenic agent into the crosslinkedpolyvinylalcohol microspheres. More preferably, the anti-angiogenicagents described in §4.1 above can be used.

This invention will be more completely described by means of thefollowing examples, which are to be considered illustrative and notlimitative.

5. EXAMPLES

Materials:

All chemical reagents including polyvinylalcohol are from Aldrich,Europe. All biological reagents such as dextran derivatives, celladhesion factor, etc. are from Sigma, U.S.A. The agitation system andthe sieving machine are from Prolabo, France.

Example 1 Preparation of Crosslinked Microspheres Comprising 5%Polyvinylalcohol

Five grams of polyvinylalcohol are dissolved in 75 ml of a 0.5 MH₂SO₄-0.1 M NaCl solution under stirring. The suspension is agitateduntil a clear solution forms and then 25 ml of formalaldehyde are addedto the solution. The resulting mixture is rapidly poured into 500 ml ofagitated paraffin oil containing 2% of sorbitan sesquioleate. Underthese conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of formaldehyde onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size is managed by the speed of agitation of the emulsion. Forexample, in order to obtain microspheres with diameter around 300 μm(average dimension), the agitation speed is kept at about 250 rpm.

Hydrogel microspheres of polyvinylalcohol are then collected byfiltration. Alternatively, hydrogel microspheres of polyvinylalcohol maybe collected by centrifugation or by simple decanting. Residue oil isextracted by non-polar solvents or chlorinated solvents such asmethylene chloride. The resulting oil-free microspheres are then treatedwith a 0.5 M Tris-HCl buffer (pH 9) overnight at room temperature toneutralize excess aldehydes.

Finally, the polyvinylalcohol microspheres are washed with physiologicalaqueous buffers, sieved to desired diameter, sterilized and stored asliquid suspensions. This material can be used for embolizationprocedure.

Example 2 Preparation of Crosslinked Microspheres Comprising 20%Polyvinylalcohol

Twenty grams of polyvinylalcohol are dissolved in 75 ml of a 0.5 MH₂SO₄-0.1 M NaCl solution under stirring. The suspension is agitateduntil a clear solution forms and then 25 ml of formalaldehyde are addedto the solution. The resulting mixture is rapidly poured into 500 ml ofagitated paraffin oil containing 2% of sorbitan sesquioleate. Underthese conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of formaldehyde onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 3 Preparation of Crosslinked Microspheres Comprising 10%Polyvinylalcohol

Ten gram of polyvinylalcohol are dissolved in 75 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms and then 25 ml of a 25% aqueous solution ofglutaraldehyde are added to the solution. The resulting mixture israpidly poured into 500 ml of agitated paraffin oil containing 2% ofsorbitan sesquioleate. Under these conditions, an emulsion is formedwith droplets of polyvinylalcohol in suspension oil. The emulsion isheated at about 80° C. for at least 6 hours to obtain the condensationof glutaraldehyde on polyvinylalcohol chains and thus forming sphericalparticles of crosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 4 Preparation of Crosslinked Microspheres Comprising 10%Polyvinylalcohol

Ten gram of polyvinylalcohol are dissolved in 85 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms and then 15 ml of a 25% aqueous solution of glyoxal areadded to the solution. The resulting mixture is rapidly poured into 500ml of agitated paraffin oil containing 2% of sorbitan sesquioleate.Under these conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of glyoxal onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 5 Preparation of Polyvinylalcohol Microspheres ContainingCollagen

Ten gram of polyvinylalcohol are dissolved in 75 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms. To this solution 10 ml of 2% collagen in water are addedunder stirring and then 15 ml of a 50% aqueous solution ofglutaraldehyde are added. The resulting mixture is rapidly poured into500 ml of agitated paraffin oil containing 2% of sorbitan sesquioleate.Under these conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of glutaraldehyde onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 6 Preparation of Polyvinyl Alcohol Microspheres Containing DEAEDextran

Ten gram of polyvinylalcohol are dissolved in 75 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms. To this solution 10 ml of 1% DEAE dextran in water areadded under stirring and then 15 ml of a 50% aqueous solution ofglutaraldehyde are added. The resulting mixture is rapidly poured into500 ml of agitated paraffin oil containing 2% of sorbitan sesquioleate.Under these conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of glutaraldehyde onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 7 Preparation of Polyvinylalcohol Microspheres Containing CMDextran

Ten gram of polyvinylalcohol are dissolved in 75 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms. To this solution 10 ml of 1% CM dextran in water areadded under stirring and then 15 ml of a 50% aqueous solution ofglutaraldehyde are added. The resulting mixture is rapidly poured into500 ml of agitated paraffin oil containing 2% of sorbitan sesquioleate.Under these conditions, an emulsion is formed with droplets ofpolyvinylalcohol in suspension oil. The emulsion is heated at about 80°C. for at least 6 hours to obtain the condensation of glutaraldehyde onpolyvinylalcohol chains and thus forming spherical particles ofcrosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 8 Preparation of Polyvinylalcohol Microspheres ContainingCollagen and DEAE Dextran

Ten gram of polyvinylalcohol are dissolved in 65 ml of a 0.5 M H₂SO₄-0.1M NaCl solution under stirring. The suspension is agitated until a clearsolution forms. To this solution 10 ml of 1% DEAE dextran in water and10 ml of 2% collagen in water are added under vigorous stirring and then15 ml of a 50% aqueous solution of glutaraldehyde are added. Theresulting mixture is rapidly poured into 500 ml of agitated paraffin oilcontaining 2% of sorbitan sesquioleate. Under these conditions, anemulsion is formed with droplets of polyvinylalcohol in suspension oil.The emulsion is heated at about 80° C. for at least 6 hours to obtainthe condensation of glutaraldehyde on polyvinylalcohol chains and thusforming spherical particles of crosslinked polyvinylalcohol.

Particle size control, microspheres collection, oil extraction,neutralization of aldehydes, microspheres wash, sieve and sterilizationare conducted as described in Example 1.

Example 9 Preparation of Polyvinylalcohol Microspheres ContainingMagnetite

Fifty ml of polyvinylalcohol microspheres obtained according to Examples1 to 8 are each packed into a 16 mm diameter chromatographic column andwashed with a physiological buffer. The column is then loaded with acolloidal suspension of ferrofluid (very small particles of magnetite)at a flow rate of 10 ml/hour. Particles of magnetite are adsorbed by thepolyvinylalcohol hydrogel network and permanently trapped. Resultingmicrospheres are used for regular embolization procedure and can bemonitored by MRI.

Example 10 Impregnated Polyvinylalcohol Microspheres With AngiogenesisInhibitors

Polyvinylalcohol microspheres obtained according to Examples 1 to 8 aredehydrated by sequential washing with ethanol to eliminate water.Ethanol is eliminated by washing with acetone and finally thepolyvinylalcohol microspheres are dehydrated under dry nitrogen. Anaqueous solution of 10 mg/ml of thalidomide is prepared and 1 gram ofdry polyvinylalcohol microspheres is mixed with 12 ml of drug solution.The suspension is gently agitated for 2 hours. Dry microspheres swellwhile adsorbing the drug in solution.

The resulting microspheres impregnated with the drug are used for anormal embolization procedure.

Example 11 Absorption of Drugs by Ion Exchange on PolyvinylalcoholMicrospheres

Polyvinylalcohol microspheres obtained according to Examples 6 and 8containing about 80 μmol of cationic groups can adsorb anionic moleculesby ion exchange. Microspheres are equilibrated with a 10 mM Tris-HClbuffer (pH 7.5) in which the molecule of interest, such asanti-angiogenic or anti-inflammatory agents, are previously dissolved.Under these conditions the molecule of interest is adsorbed by ionexchange effect, and the resulting microspheres can be used for regularembolization procedures.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. Microspheres useful for embolization, whereinsaid microspheres comprise crosslinked polyvinyl alcohol, have adiameter ranging from about 50 μm to about 1,000 μm, and aresubstantially spherical and sterile, wherein the difference in diameterbetween the microspheres is from about 0 μm to about 150 μm, wherein themicrospheres comprise from about 0.5% to about 20% polyvinyl alcohol byweight in hydrogel form.
 2. The microspheres of claim 1, wherein saidmicrospheres further comprise a cell adhesion promoter.
 3. Themicrospheres of claim 1, wherein said microspheres further comprise amarking agent.
 4. The microspheres of claim 3, wherein the marking agentis selected from the group consisting of a dye, an imaging agent and acontrasting agent.
 5. The microspheres of claim 1, further comprising ananti-angiogenic agent.
 6. The microspheres of claim 1, wherein themicrospheres are substantially uniform in size.
 7. The microspheres ofclaim 1, wherein the microspheres are substantially uniform in shape. 8.Microspheres useful for embolization, wherein said microspheres comprisecrosslinked polyvinyl alcohol, have a diameter ranging from about 10 μmto about 2,000 μm, and are substantially spherical and sterile, whereinthe microspheres comprise from about 0.5% to about 20% polyvinyl alcoholby weight in hydrogel form, wherein the surface of the microspheresappears smooth under less than 1000-fold magnification and saidmicrospheres do not aggregate by surface interaction, and wherein themicrospheres are configured to occlude blood flow through a vessel whenpositioned in the vessel.
 9. An injectable sterile suspension comprisingthe microspheres of claim 1 and a suitable liquid carrier.
 10. A kitcomprising the microspheres of claim
 1. 11. A method for the treatmentof a cancer or tumor in a mammal by embolization, comprisingadministering to the mammal an effective amount of the microspheres ofclaim
 1. 12. The method of claim 11, wherein the cancer or tumor is ahepatocellular carcinoma, liver metastases, uterine tumor, uterinefibroid, colorectal cancer, bone metastases, melanomas, cancer of thehead, cancer of the neck, or intracranial meningioma.
 13. A method forthe treatment of a small intestinal hemorrhage in a mammal byembolization, comprising administering to the mammal an effective amountof the microspheres of claim
 1. 14. A method for the treatment of avascular malformation in a mammal by embolization, comprisingadministering to the mammal an effective amount of the microspheres ofclaim
 1. 15. The method of claim 14, wherein the vascular malformationis an arteriovenous malformation (AVM).
 16. The microspheres of claim 2,wherein the cell adhesion promoter is selected from at least one of thefollowing: carboxymethyl (CM) dextran, collagen, DEAE dextran, gelatin,glucosaminoglycans, fibronectin, lectins, and polycations.
 17. Themicrospheres of claim 16, wherein the cell adhesion promoter is selectedfrom at least one of the following: carboxymethyl (CM) dextran,collagen, and DEAE dextran.
 18. The microspheres of claim 8, wherein thedifference in diameter between the microspheres is from about 0 μm toabout 150 μm.